Method for predicting and evaluating therapeutic effect in diseases related to il-6 and neutrophils

ABSTRACT

It has become clear that the therapeutic effect of an IL-6 inhibitor for IL-6- and neutrophil-associated diseases can be predicted using the expression level of neutrophil-associated genes as an indicator. It has also become clear that an IL-6 inhibitor is effective for the treatment of IL-6- and neutrophil-associated diseases in patients with high expression levels of neutrophil-associated genes. The present invention provides a method for selecting cases of IL-6- and neutrophil-associated diseases in which treatment with an IL-6 inhibitor is effective, as well as a method for effectively treating patients with IL-6- and neutrophil-associated diseases and with high expression levels of neutrophil-associated genes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 16/609,053, 371(c) date Oct. 28, 2019, which is a U.S. National Phase of PCT Application No. PCT/JP2018/017374, filed May 1, 2018, which claims the benefit of Japanese Patent Application No. 2017-091600, filed May 2, 2017, each of which is incorporated herein by reference in its entirety.

Reference to Sequence Listing Submitted Electronically

The content of the electronically submitted sequence listing (Name: 6663_0239_Sequence_Listing.xml; Size: 36.4 kilobytes; and Date of Creation: Jun. 22, 2023) filed with the application is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates to methods for predicting the therapeutic effect of an IL-6 inhibitor in patients with IL-6- and neutrophil-associated diseases (may be referred to as neutrophil-associated diseases hereinbelow). The present invention also relates to methods for determining the therapeutic effect of an IL-6 inhibitor in patients with neutrophil-associated diseases. Furthermore, the present invention relates to agents for treating neutrophil-associated diseases comprising an IL-6 inhibitor, and particularly to agents for treating neutrophil-associated diseases which are administered to patients with high expression levels of neutrophil-associated genes. In addition, the present invention also relates to methods for treating neutrophil-associated diseases, and the methods are characterized in that an IL-6 inhibitor is administered to patients with high expression levels of neutrophil-associated genes.

BACKGROUND ART

Diseases in which IL-6 is associated with their pathological conditions (IL-6-associated diseases), such as neuromyelitis optica (NMO), rheumatoid arthritis, and juvenile idiopathic arthritis, are known as diseases that are treatable with interleukin-6 (IL-6) inhibitors (Non-patent Documents 1 and 2). As IL-6 inhibitors, ingredients such as the following are utilized (Patent Document 1):

-   -   Anti-IL-6 antibodies,     -   Anti-IL-6 receptor (IL-6R) antibodies,     -   Signal transducer and activator of transcription 3 (STATS)         inhibitors, and Janus kinase-1 (JAK1) inhibitors.

Among these known IL-6 inhibitors, for example, tocilizumab (TCZ; generic name), an anti-IL-6R antibody, has been approved as a therapeutic drug for Castleman's disease, rheumatoid arthritis, and juvenile idiopathic arthritis.

Meanwhile it is known that one of the actions of tocilizumab is to decrease or increase the number of neutrophils. Neutrophils have been reported to be associated with the pathological condition of neuromyelitis optica (NMO) (Non-patent Documents 3 and 4). In addition to NMO, Neuro-Sweet disease, stroke, cerebral infarction, and such are known as diseases in which IL-6 and neutrophils are associated with their pathological conditions (IL-6- and neutrophil-associated diseases; Non-patent Document 5). However, it is not known how tocilizumab affects the viability and function of neutrophils, and further elucidation of the mechanism of action of IL-6 inhibitors, represented by anti-IL-6R antibodies, is desired.

CITATION LIST Patent Document

-   [Patent Document 1] WO2010/035769

Non-Patent Document

-   [Non-patent Document 1] Neurology 2014; 82; 1302-1306 -   [Non-patent Document 2] Annu.Rev.Pharmacol. Toxicol.2012.52:199-219 -   [Non-patent Document 3] Multiple Sclerosis Journal, 18(12),     1801-1803, 2012 -   [Non-patent Document 4] ANN NEUROL 2012; 71: 323-333 -   [Non-patent Document 5] Clinical Neurology 2012; 52:1234-1236

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An objective of the present invention is to provide a new strategy for treating neutrophil-associated diseases by elucidating the mechanism of action of IL-6 inhibitors.

Means for Solving the Problems

The present inventors conducted intense research to solve the above-mentioned problems. Specifically, they first administered tocilizumab, an IL-6 inhibitor, to neuromyelitis optica patients as subjects, and analyzed by DNA microarray genes that were highly expressed compared to healthy individuals prior to administration and that reduced expression after administration. The results revealed that among the genes with greater fluctuation in expression level, the top nine genes are related to the granule content of neutrophils. It has not been known until now that inhibiting IL-6R alters genes associated with neutrophils (referred to as neutrophil-associated genes hereinbelow). Based on this finding, the present inventors have found that the expression levels of the neutrophil-associated genes can be used as an indicator for predicting or determining the therapeutic effect of IL-6 inhibitors for neutrophil-associated diseases. The present inventors have also found that IL-6 inhibitors are useful for the treatment of neutrophil-associated diseases.

The present invention is based on such findings and specifically relates to the following inventions.

-   -   [1] A method for predicting the therapeutic effect of an IL-6         inhibitor in a patient with an IL-6- and neutrophil-associated         disease, comprising:         -   (i) measuring the expression level of a             neutrophil-associated gene in a sample obtained from the             patient with an IL-6- and neutrophil-associated disease, and         -   (ii) comparing the expression level measured in step (i)             with a control, wherein the therapeutic effect of the IL-6             inhibitor is indicated to be high when the expression level             is higher than the control.     -   [2] The method of [1], wherein the control is the expression         level of the neutrophil-associated gene in a sample obtained         from a healthy individual.     -   [3] The method of [1], wherein in step (ii), the therapeutic         effect of the IL-6 inhibitor is indicated to be high when the         expression level measured in step (i) is five times or higher         than the expression level of the neutrophil-associated gene in a         sample obtained from a healthy individual.     -   [4] The method of [1], wherein in step (ii), the therapeutic         effect of the IL-6 inhibitor is indicated to be high when the         expression level measured in step (i) is ten times or higher         than the expression level of the neutrophil-associated gene in a         sample obtained from a healthy individual.     -   [5] A method for determining the therapeutic effect of an IL-6         inhibitor in a patient with an IL-6- and neutrophil-associated         disease, comprising:         -   (i) measuring the expression level of a             neutrophil-associated gene in a sample obtained from the             patient with an IL-6- and neutrophil-associated disease who             has been administered with the IL-6 inhibitor, and         -   (ii) comparing the expression level measured in step (i)             with a control, wherein the therapeutic effect of the IL-6             inhibitor is indicated to be high when the expression level             is lower than the control.     -   [6] The method of [5], wherein the control is the expression         level of the neutrophil-associated gene in a sample obtained         from the patient prior to administration of the IL-6 inhibitor.     -   [7] The method of [5], wherein in step (ii), the therapeutic         effect of the IL-6 inhibitor is indicated to be high when the         expression level measured in step (i) is less than 0.5 times the         expression level of the neutrophil-associated gene in a sample         obtained from the patient prior to administration of the IL-6         inhibitor.     -   [8] The method of [5], wherein in step (ii), the therapeutic         effect of the IL-6 inhibitor is indicated to be high when the         expression level measured in step (i) is less than 0.2 times the         expression level of the neutrophil-associated gene in a sample         obtained from the patient prior to administration of the IL-6         inhibitor.     -   [9] The method of any one of [1] to [8], wherein the         neutrophil-associated gene is at least one gene selected from         the group consisting of CTSG (cathepsin G), DEFA4 (defensin,         alpha 4, corticostatin), AZU1 (azurocidin 1), BPI         (bactericidal/permeability-increasing protein), ELANE (elastase,         neutrophil expressed), LTF (lactotransferrin), DEFA3 (defensin,         alpha 3, neutrophil-specific), LCN2 (lipocalin 2), and CAMP         (cathelicidin antimicrobial peptide).     -   [10] The method of any one of [1] to [9], wherein the         neutrophil-associated gene is at least one gene selected from         the group consisting of CTSG (cathepsin G), DEFA4 (defensin,         alpha 4, corticostatin), and AZU1 (azurocidin 1).     -   [11] The method of any one of [1] to [10], wherein the IL-6         inhibitor is an anti-IL-6 receptor antibody.     -   [12] Use of a neutrophil-associated gene in predicting or         determining the therapeutic effect of an IL-6 inhibitor for an         IL-6- and neutrophil-associated disease.     -   [13] A therapeutic agent comprising an IL-6 inhibitor as an         active ingredient, which is for administration to a patient in         whom treatment with the IL-6 inhibitor has been indicated to be         highly effective by the method of [1].     -   [14] A therapeutic agent comprising an IL-6 inhibitor as an         active ingredient, which is for continued administration to a         patient in whom treatment with the IL-6 inhibitor has been         indicated to be highly effective by the method of [5].     -   [15] The therapeutic agent of or [14], wherein the IL-6         inhibitor is an anti-IL-6 receptor antibody.     -   [16] The therapeutic agent of [15], wherein the anti-IL-6         receptor antibody is a chimeric antibody, a humanized antibody,         or a human antibody.     -   [17] The therapeutic agent of [13], which is characterized in         that it is administered to a patient in whom treatment with the         IL-6 inhibitor has been judged to be highly effective by the         method of any one of [1] to [11].     -   [18] A method for treating an IL-6- and neutrophil-associated         disease, comprising administering an IL-6 inhibitor to a patient         suffering from the IL-6- and neutrophil-associated disease,         wherein a sample obtained from the patient shows high expression         level of a neutrophil-associated gene.     -   [19] A method for treating an IL-6- and neutrophil-associated         disease, comprising administering an IL-6 inhibitor to a patient         in whom treatment with the IL-6 inhibitor has been judged to be         highly effective by the method of any one of [1] to [11].     -   [20] An IL-6 inhibitor for use in the treatment of an IL-6- and         neutrophil-associated disease, which is administered to a         patient suffering from the IL-6- and neutrophil-associated         disease, wherein a sample obtained from the patient shows high         expression level of a neutrophil-associated gene.     -   [21] Use of an IL-6 inhibitor in the production of an agent for         treating an IL-6- and neutrophil-associated disease, wherein the         therapeutic agent is administered to a patient suffering from         the IL-6- and neutrophil-associated disease, and wherein a         sample obtained from the patient shows high expression level of         a neutrophil-associated gene.     -   [22] Use of an IL-6 inhibitor in the treatment of an IL-6- and         neutrophil-associated disease, which is characterized in that         the expression level of a neutrophil-associated gene in a sample         obtained from a patient receiving the treatment is high.     -   [23] A method for the manufacture of an agent for treating an         IL-6- and neutrophil-associated disease comprising an IL-6         inhibitor as an active ingredient, wherein the therapeutic agent         is administered to a patient suffering from the IL-6- and         neutrophil-associated disease, and wherein a sample obtained         from the patient shows high expression level of a         neutrophil-associated gene.     -   [24] A kit for predicting or determining the therapeutic effect         of an IL-6 inhibitor in a patient suffering from an IL-6- and         neutrophil-associated disease, which is characterized in that it         comprises a reagent for measuring the expression level of a         neutrophil-associated gene.     -   [25] A reagent for detecting a neutrophil-associated gene for         use in predicting or determining the therapeutic effect of an         IL-6 inhibitor in a patient suffering from an IL-6- and         neutrophil-associated disease.     -   [26] Use of a reagent for detecting a neutrophil-associated gene         in the manufacture of an agent for predicting or determining the         therapeutic effect of an IL-6 inhibitor in a patient suffering         from an IL-6- and neutrophil-associated disease.     -   [27] Use of a reagent for detecting a neutrophil-associated gene         in predicting or determining the therapeutic effect of an IL-6         inhibitor in a patient suffering from an IL-6- and         neutrophil-associated disease.     -   [28] A method for detecting a marker for predicting or a marker         for determining the therapeutic effect of an IL-6 inhibitor in a         patient suffering from an IL-6- and neutrophil-associated         disease, comprising measuring the expression level of a         neutrophil-associated gene in a sample obtained from the         patient.     -   [29] A method for classifying a patient suffering from an IL-6-         and neutrophil-associated disease, comprising determining that a         patient suffering from an IL-6- and neutrophil-associated         disease, the expression level of a neutrophil-associated gene in         a sample obtained from the patient being high, can be treated         with high efficacy with an IL-6 inhibitor.     -   [30] The method, use, therapeutic agent, or kit of any one of         [1] to [29], wherein the IL-6- and neutrophil-associated disease         is neuromyelitis optica, Neuro-Sweet disease, stroke, or         cerebral infarction.     -   [31] The method, use, therapeutic agent, or kit of any one of         [1] to [30], wherein the IL-6- and neutrophil-associated disease         is neuromyelitis optica.

Effects of the Invention

The present invention provides a method for predicting and determining the therapeutic effect of an IL-6 inhibitor in a neutrophil-associated disease using the expression level of a neutrophil-associated gene as an indicator. The method of the present invention allows one to avoid administering an IL-6 inhibitor to patients for whom therapeutic effect of the IL-6 inhibitor cannot be expected or patients who will be forced to experience severe side effects with an IL-6 inhibitor, and allows one to select the appropriate treatment regimen. In addition, the present invention shows that an IL-6 inhibitor is efficacious in treating a patient with a neutrophil-associated disease who has elevated level of expression of a neutrophil-associated gene. Thus, the present invention provides an agent and a method for treating a neutrophil-associated disease with high expression level of a neutrophil-associated gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Venn diagram showing the number of differentially expressed probes in each category.

FIG. 2 shows RT-PCR results confirming the whole blood mRNA levels of CTSG, DEFA4, and AZU1 in NMO patients before and after TCZ treatment. The vertical axis shows the relative expression intensity in relation to the expression level of ACTB.

MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below.

The present invention relates to genetic markers for predicting and/or determining the therapeutic effect of an IL-6 inhibitor in a patient with a neutrophil-associated disease (markers for determining whether or not treatment with an IL-6 inhibitor for a neutrophil-associated disease can be applied; also referred to as genetic markers of the present invention hereafter). In other words, the present invention relates to use of the genetic markers of the present invention in predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease. The genetic markers of the present invention can be used to predict and/or determine that a patient with neutrophil-associated disease can be treated with high efficacy with an IL-6 inhibitor. Thus, the genetic markers of the present invention (or the marker genes of the present invention) can also be referred to as neutrophil-associated genes, genes associated with neutrophils and IL-6, or neutrophil/IL-6 associated genes. Genetic markers of the present invention may include, for example, the following genes:

-   -   CTSG (cathepsin G),     -   DEFA4 (defensin, alpha 4, corticostatin),     -   AZU1 (azurocidin 1),     -   BPI (bactericidal/permeability-increasing protein),     -   ELANE (elastase, neutrophil expressed),     -   LTF (lactotransferrin),     -   DEFA3 (defensin, alpha 3, neutrophil-specific),     -   LCN2 (lipocalin 2), and     -   CAMP (cathelicidin antimicrobial peptide).

One marker may be selected and used from among the markers listed herein as a marker of the present invention, and two or more markers may be selected and used in combination. That is, the neutrophil-associated gene in the present invention may be at least one gene selected from the group consisting of CTSG, DEFA4, AZU1, BPI, ELANE, LTF, DEFA3, LCN2, and CAMP. These neutrophil-associated genes can be any one, or two or more, preferably three or more, more preferably five or more, and all nine of them in a preferred embodiment.

The nucleic acid sequence and amino acid sequence of each of the marker genes listed here as examples of neutrophil-associated genes in humans are known as below.

Nucleic acid sequence Amino acid sequence CTSG (cathepsin G; NM_001911) SEQ ID NO: 1 SEQ ID NO: 2 DEFA4 (defensin, alpha 4, corticostatin; NM_001925) SEQ ID NO: 3 SEQ ID NO: 4 AZU1 (azurocidin 1; NM_001700) SEQ ID NO: 5 SEQ ID NO: 6 BPI (bactericidal/permeability-increasing protein; SEQ ID NO: 7 SEQ ID NO: 8 NM_001725) ELANE (elastase, neutrophil expressed; NM_001972) SEQ ID NO: 9 SEQ ID NO: 10 LTF (lactotransferrin; NM_002343) SEQ ID NO: 11 SEQ ID NO: 12 DEFA3 (defensin, alpha 3, neutrophil-specific; NM_005217) SEQ ID NO: 13 SEQ ID NO: 14 LCN2 (lipocalin 2; NM_005564) SEQ ID NO: 15 SEQ ID NO: 16 CAMP (cathelicidin antimicrobial peptide; NM_004345) SEQ ID NO: 17 SEQ ID NO: 18

The structures of the homologs of various neutrophil-associated genes in non-human species, used as genetic markers, have also been revealed. Therefore, homologs of each gene can be selected depending on the animal species for evaluation in non-human model animals.

Diseases for which the therapeutic effect of an IL-6 inhibitor can be predicted/determined using the genetic markers of the present invention as indicators are diseases in which neutrophils are associated with their clinical conditions and diseases in which IL-6 is associated with theirs clinical conditions (referred to as “IL-6- and neutrophil-associated diseases” or “neutrophil/IL-6-associated diseases”, and hereafter sometimes referred to as neutrophil-associated diseases). The diseases are characterized by high expression levels of genetic markers of the present invention (or neutrophil-associated genes, genes associated with neutrophils and IL-6, neutrophil/IL-6-associated genes). Examples of such diseases include, but are not limited to, neuromyelitis optica (NMO), Neuro-Sweet disease, stroke, cerebral infarction, and such. In the present invention, the term “neuromyelitis optica” includes neuromyelitis optica spectrum disorders.

Markers in the present invention refer to specific chemical substances in a living body that can also be rephrased as biomarkers, and they can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responsiveness to treatments. Markers are useful for assessment of the presence of, progression of, or susceptibility to a disease, for evaluation or prediction of the effect, optimal dosage, or safety of a drug, for prediction of prognosis, and such. Markers in the present invention are specified by their gene names It is preferable to measure the marker genes as polypeptides or polynucleotides, and it is particularly preferable to measure them as polynucleotides.

The present invention relates to methods for predicting the therapeutic effect of an IL-6 inhibitor in a patient with a neutrophil-associated disease, which comprise measuring the expression level of a neutrophil-associated gene in a sample obtained from the patient suffering from a neutrophil-associated disease. The neutrophil-associated gene is preferably at least one gene selected from the group consisting of CTSG, DEFA4, AZU1, BPI, ELANE, LTF, DEFA3, LCN2, and CAMP.

In the above methods for predicting therapeutic effect of the present invention, the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease is predicted to be high when the expression level of a genetic marker of the present invention in a patient with the neutrophil-associated disease is high. Thus, the methods of the present invention may further comprise a step of predicting that the therapeutic effect of an IL-6 inhibitor is high for a patient, a sample obtained from whom has a high expression level of a genetic marker of the present invention.

The present invention also relates to methods for determining the therapeutic effect of an IL-6 inhibitor in a patient with a neutrophil-associated disease, which comprise measuring the expression level of a neutrophil-associated gene in a sample obtained from the patient suffering from a neutrophil-associated disease who has been administered with the IL-6 inhibitor. The neutrophil-associated gene is preferably at least one gene selected from the group consisting of CTSG, DEFA4, AZU1, BPI, ELANE, LTF, DEFA3, LCN2, and CAMP.

In the above methods for determining therapeutic effect of the present invention, when the expression level of a genetic marker of the present invention in a patient with neutrophil-associated disease is reduced by administration of an IL-6 inhibitor, the therapeutic effect of the IL-6 inhibitor is determined to be high in this patient. The biological sample used to measure the expression level of a genetic marker of the present invention for determining the therapeutic effect of an IL-6 inhibitor is not particularly limited as long as it is a sample taken from a patient who has been administered with the IL-6 inhibitor and can be used in the determination. The sample is, for example, a sample obtained from a patient one month, two months, several months, one year, two years, or several years after administration of the IL-6 inhibitor.

The present invention relates to a method for predicting the therapeutic effect of an IL-6 inhibitor in a patient with a neutrophil-associated disease, which comprises

-   -   (i) measuring the expression level of a neutrophil-associated         gene in a sample obtained from the patient with a         neutrophil-associated disease, and     -   (ii) comparing the expression level measured in step (i) with a         control, wherein the therapeutic effect of the IL-6 inhibitor is         indicated to be high when the expression level is higher than         the control.

Alternatively, the present invention can further comprise after (ii), a step of

-   -   (iii) administering the IL-6 inhibitor to the patient with a         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

That is, the present invention relates to a method for treating a neutrophil-associated disease comprising the steps of (i)-(iii).

Alternatively, the present invention relates to the use of a reagent for detecting the expression level of a neutrophil-associated gene in the manufacture of an agent for predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease.

Alternatively, the present invention relates to the use of a reagent for detecting the expression level of a neutrophil-associated gene in predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease.

Alternatively, the present invention relates to the use of a reagent for detecting the expression level of a neutrophil-associated gene in predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the IL-6 inhibitor is administered to the patient with the         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

The present invention also relates to the use of an IL-6 inhibitor in the treatment of a neutrophil-associated disease, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the IL-6 inhibitor is administered to the patient with the         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

Alternatively, the present invention relates to the use of a reagent for detecting the expression level of a neutrophil-associated gene in the manufacture of an agent for predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the IL-6 inhibitor is administered to the patient with the         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

The present invention also relates to the use of an IL-6 inhibitor in the manufacture of an agent for treating a neutrophil-associated disease, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the IL-6 inhibitor is administered to the patient with the         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

Alternatively, the invention relates to a method for detecting a marker for predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease, which comprises measuring the expression level of a neutrophil-associated gene in a sample obtained from a patient with the neutrophil-associated disease.

Alternatively, the invention relates to an IL-6 inhibitor or an agent for treating a neutrophil-associated disease, for use in the administration to a patient with a neutrophil-associated disease in whom treatment with the IL-6 inhibitor has been indicated to be highly effective by a method comprising the steps below, or for use in the treatment of a neutrophil-associated disease for which treatment with the IL-6 inhibitor has been indicated to be highly effective by a method comprising the steps below:

-   -   (i) measuring the expression level of a neutrophil-associated         gene in a sample obtained from the patient with a         neutrophil-associated disease, and     -   (ii) indicating that the therapeutic effect of the IL-6         inhibitor is high when the expression level of the         neutrophil-associated gene is high compared with that of a         healthy individual or when the expression level of the         neutrophil-associated gene in the patient after administration         of the IL-6 inhibitor is low compared with that in the patient         before administration of the IL-6 inhibitor.

Alternatively, the present invention can further comprise after (ii), a step of

-   -   (iii) administering an IL-6 inhibitor or an agent for treating a         neutrophil-associated disease to the patient with a         neutrophil-associated disease in whom treatment with the IL-6         inhibitor has been indicated to be highly effective.

Alternatively, the present invention relates to an agent for treating a neutrophil-associated disease comprising an IL-6 inhibitor as an active ingredient, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the agent for treating a neutrophil-associated disease is         for administration to a patient with the neutrophil-associated         disease in whom treatment with the IL-6 inhibitor has been         indicated to be highly effective.

Alternatively, the present invention relates to an agent for predicting or determining the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease, comprising a reagent for detecting the expression level of a neutrophil-associated gene, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the predictor or determinant is for administering the IL-6         inhibitor to the patient with the neutrophil-associated disease         in whom treatment with the IL-6 inhibitor has been indicated to         be highly effective.

In the present invention, as the reagent for detecting the expression level of a neutrophil-associated gene, a reagent for detecting an mRNA encoding the gene to be detected or a translation product thereof (i.e., a protein) can be used. The translation product can be detected by using the amount of the protein or its biological activity as an indicator. Specifically, oligonucleotides that specifically bind to mRNA are among the typical detection reagents. Oligonucleotides can detect mRNA by their specific binding. In a known technique such as PCR, the mRNA to be detected can be amplified, and the mRNA can be detected using the amplification product as an indicator. Alternatively, antibodies that specifically bind to a protein which is a translation product are preferred detection reagents. Antibodies are useful for immunoassays and Western blot analysis. These detection reagents may be appropriately labelled or conjugated to a solid phase and applied to various assay formats.

Alternatively, the present invention also provides a kit for detecting a marker for predicting or determining therapeutic effect in a neutrophil-associated disease, comprising:

-   -   (i) a reagent for detecting the expression level of a         neutrophil-associated gene in a sample, and     -   (ii) a positive control sample for the expression level of the         neutrophil-associated gene.

The positive control sample is not particularly limited as long as the amount of the marker contained in a sample is preliminarily specified, and it can be appropriately prepared depending on the form of the marker measured by the kit. For example, when the form of the marker is a polypeptide, a sample containing polypeptides that are the same as the marker and have been isolated, purified, and quantified, is preferred as a positive control sample.

The kit of the present invention is a kit for detecting a marker for predicting or determining therapeutic effect in a neutrophil-associated disease, wherein

-   -   (i) the expression level of a neutrophil-associated gene in a         sample obtained from a patient with the neutrophil-associated         disease is measured,     -   (ii) a high expression level of the neutrophil-associated gene         compared with that of a healthy individual, or a low expression         level of the neutrophil-associated gene in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor indicates         that the therapeutic effect of the IL-6 inhibitor is high, and     -   (iii) the kit is for administering the IL-6 inhibitor to the         patient with the neutrophil-associated disease in whom treatment         with the IL-6 inhibitor has been indicated to be highly         effective.

In the present invention, a positive control sample is a sample comprising at least one transcription product (mRNA) or translation product (protein) of a neutrophil-associated gene in an amount that provides a level detected in a group of patients in whom treatment with an IL-6 inhibitor was highly effective. It is preferable that the positive control sample is the same as the sample actually taken from the patient for measurement. Therefore, when the target is a blood sample such as whole blood, the preferred positive control sample is also a blood sample. Blood samples include whole blood, serum, and plasma. The positive control can be liquid or lyophilized In the case of a liquid sample, it may be concentrated.

The method for predicting the therapeutic effect of an IL-6 inhibitor in a patient suffering from a neutrophil-associated disease of the present invention can be rephrased as a method for evaluating the efficacy of an IL-6 inhibitor treatment in a patient suffering from a neutrophil-associated disease. It can also be rephrased as a method for selecting a patient who is suitable for an IL-6 inhibitor treatment from among patients suffering from neutrophil-associated diseases. Alternatively, it can also be rephrased as a method for detecting a marker for predicting the therapeutic effect of an IL-6 inhibitor or a marker for evaluating efficacy of the treatment in a patient suffering from a neutrophil-associated disease, or a marker for selecting a patient suitable for the treatment. These methods can be performed in vitro using samples obtained from patients.

In the present invention, the method for predicting the therapeutic effect can be rephrased as a method for predicting prognosis, a method for determining whether or not the treatment is applicable, a method for diagnosing the therapeutic effect, a method for deciding whether or not to continue the treatment, and such.

Also, in the present invention, the expression “the therapeutic effect is indicated to be high” can be rephrased as “the therapeutic effect is judged/determined to be high”.

Also, “a patient with a neutrophil-associated disease in whom treatment with an IL-6 inhibitor has been indicated to be highly effective” in the present invention can be rephrased as “a patient suitable for IL-6 inhibitor therapy”, “a patient responsive to IL-6 inhibitor therapy”, or such. Therefore, the present invention relates to a method for identifying a patient suitable for treatment of a neutrophil-associated disease with an IL-6 inhibitor, comprising:

-   -   (i) measuring the expression level of a neutrophil-associated         gene in a sample obtained from a patient with the         neutrophil-associated disease, and     -   (ii) indicating that the therapeutic effect of the IL-6         inhibitor is high when the expression level of the         neutrophil-associated gene is high compared to that in a healthy         individual, or when the expression level of the         neutrophil-associated gene is low in the patient after         administration of the IL-6 inhibitor compared with that in the         patient before administration of the IL-6 inhibitor.

The patient from whom the sample is obtained in the present invention may be any patient suffering from a neutrophil-associated disease. It may be a patient who has not yet been treated for a neutrophil-associated disease or a patient who is already receiving treatment. In the case of a patient already receiving treatment with an IL-6 inhibitor, the present invention provides a method for monitoring the responsiveness of the patient to an IL-6 inhibitor treatment, or a method for deciding whether or not to continue an IL-6 inhibitor treatment for the patient.

In the present invention, a high expression level of a marker means that the measured value of the marker is higher than the predetermined value set for that marker, and a low expression level of a marker means that the measured value of the marker is below the predetermined value set for that marker. Thus, in the present method for predicting therapeutic effect, when the expression level of a marker measured in a sample obtained from a patient is higher than the predetermined value set for the marker, the therapeutic effect of an IL-6 inhibitor is indicated to be high for the patient. In one embodiment, the method for predicting therapeutic effect of the present invention may include a step of comparing the expression level of a marker measured in a sample obtained from a patient with the predetermined value set for the marker, and a step of determining that the therapeutic effect of an IL-6 inhibitor is high for the patient when the measured expression level is higher than the predetermined value. Meanwhile, in the present method for determining therapeutic effect, when the expression level of a marker measured in a sample obtained from a patient who has been administered with an IL-6 inhibitor is lower than the predetermined value set for the marker, the therapeutic effect of the IL-6 inhibitor is indicated to be high for the patient. In one embodiment, the method for determining therapeutic effect of the present invention may include a step of comparing the expression level of a marker measured in a sample obtained from a patient who has been administered with an IL-6 inhibitor with the predetermined value set for the marker, and a step of determining that the therapeutic effect of the IL-6 inhibitor is high for the patient when the measured expression level is lower than the predetermined value.

The predetermined value in the present invention refers to a value determined in advance on the basis of some scientific evidence, and it may be any value as long as the value can be used as a baseline to determine whether the therapeutic effect of an IL-6 inhibitor is high or low in a patient suffering from a neutrophil-associated disease. A predetermined value in the present invention may be set for each marker.

The predetermined value in the present invention can be set from, for example, the measured value (control value) of a marker in a sample obtained from a healthy individual (control sample). Since it has already been found from the present research results that the measured value of a marker of the present invention is increased in a patient suffering from a neutrophil-associated disease compared to that in a healthy individual, a possible means may be setting the average value of the measured values of the marker in samples obtained from multiple healthy individuals as it is as the predetermined value, and another possible means may be adding a value of 1.0, 1.5, 2.0, 2.5, or 3.0 times the standard deviation to the average value of the measured values and setting it as the predetermined value. Alternatively, a value of 1.2, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 times or higher of the average value of the measured values of the marker in samples obtained from multiple healthy individuals may be set as the predetermined value. Thus, in one embodiment of the present method for predicting therapeutic effect, a higher expression level of the marker measured in a sample obtained from a patient suffering from a neutrophil-associated disease (e.g., 1.2, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 times or higher, preferably 5.0 times or more, and more preferably 10 times or more compared to the control) compared to the expression level of the marker measured in a sample obtained from a healthy individual (control sample) indicates that the therapeutic effect of an IL-6 inhibitor is high in the patient.

The predetermined value in the present invention can be set from, for example, the measured value (control value) of a marker in a sample obtained from a patient prior to administration of an IL-6 inhibitor (control sample). It has already been found from the present research results that the measured value of a marker of the present invention is decreased after administration of an IL-6 inhibitor in patients suffering from a neutrophil-associated disease compared to that prior to administration of the IL-6 inhibitor. Thus, in one embodiment, the measured value of a marker in a sample obtained from a patient prior to administration of an IL-6 inhibitor, or the average value of the measured values of the marker in samples obtained from multiple patients may be set as it is as the predetermined value. In another embodiment, a value obtained by adding a value of 1.0, 1.5, 2.0, 2.5, or 3.0 times the standard deviation to the average value of the measured values may be set as the predetermined value. Alternatively, a value of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 times or less of the measured values of a marker in samples obtained from multiple healthy individuals (or their average value) may be set as the predetermined value. Thus, in one embodiment of the present method for determining therapeutic effect, a lower expression level of a marker measured in a sample obtained from a patient after the administration of an IL-6 inhibitor (e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 times or less, preferably less than 0.5 times, and even preferably less than 0.2 times of the control) compared to the expression level of the marker measured in a sample obtained from the patient prior to administration of the IL-6 inhibitor (control sample) indicates that the IL-6 inhibitor has a high therapeutic effect in this patient.

The predetermined value in the present invention may also be set based on the results of clinical trials and such, in which multiple patients suffering from a neutrophil-associated disease were treated with an IL-6 inhibitor. When there are differences in the therapeutic effect of an IL-6 inhibitor between the group of patients in which the measured value of a marker is higher than a baseline value and the group of patients in which the measured value is lower than the baseline value, the baseline value may be used as the predetermined value in the present invention. In such a case, it is preferred that there is a statistically significant difference in therapeutic effect between the two groups. For example, in one embodiment of the present method for predicting therapeutic effect, it is indicated that the therapeutic effect of an IL-6 inhibitor is high for a patient when the expression level of a marker measured in a sample obtained from that patient, suffering from a neutrophil-associated disease, is higher compared with the expression level of the marker measured in sample(s) obtained from another patient(s) suffering from a neutrophil-associated disease for whom therapeutic effect of the IL-6 inhibitor was low. Meanwhile, in one embodiment of the present method for determining therapeutic effect, it is indicated that the therapeutic effect of an IL-6 inhibitor is high for a patient when the expression level of a marker measured in a sample obtained from that patient, suffering from a neutrophil-associated disease, is lower compared to the expression level of the marker measured in sample(s) obtained after administration of the IL-6 inhibitor from another patient(s) suffering from a neutrophil-associated disease for whom therapeutic effect of the IL-6 inhibitor was high.

The measured value and predetermined value of a marker in the present invention implies a measurement result of the expression level of a marker which has been expressed as numeric values in some way, and the value obtained from the measurement (e.g., color development intensity, etc.) may be used as it is, or a positive control sample with a known amount of the contained marker may be separately prepared and a value obtained by converting the measurement result in comparison therewith (e.g., concentration) may be used. Alternatively, the values obtained as described above may be made into scores by separating them into certain ranges (e.g., grades 1, 2, 3) and such values may be used.

Measurement of the expression level of a marker can be performed by selecting an appropriate method depending on the form of the marker or the type of sample in which the expression level of the marker is to be measured. When the form of the marker is a polypeptide, the expression level can be assessed by immunological techniques using antibodies that specifically bind the polypeptide. Immunological techniques may include, for example, enzyme-linked immunosorbent assay (ELISA, EIA), fluorescence immunoassay (FIA), radioimmunoassay (RIA), luminescent immunoassay (LIA), electrochemiluminescence (ECL), Western blotting, surface plasmon resonance, antibody array-based methods, immunohistochemical staining, fluorescence-activated cell sorting (FACS), immunochromatography, immunoprecipitation, immunonephelometry, latex-agglutination, and such.

On the other hand, when the form of the marker is a polynucleotide, it is preferable to perform measurements by genetic engineering techniques using oligonucleotides that specifically bind to the polynucleotide, and such techniques can include, for example, polymerase chain reaction (PCR), reverse-transcription PCR (RT-PCR), real-time quantitative PCR (Q-PCR), Northern blotting, and hybridization (including methods using oligonucleotide arrays such as DNA microarrays).

Measurement of the expression level of a neutrophil-associated gene in the present invention includes both polypeptide measurement and polynucleotide measurement. The expression level of a gene can generally be assessed by quantifying the expression product of the gene. Thus, the polypeptide measured in the present invention can be a protein normally encoded by a gene. A protein can be a protein that underwent posttranslational modification or a fragment of a protein, as long as it can reflect the quantitative level in a biological sample. Meanwhile, when measuring polynucleotides, it is common to measure mRNAs as an expression product of a gene. The expression levels of mRNA can also be compared per cell by correction for the expression level of a housekeeping gene.

A sample in the present invention can be rephrased as a biological sample and refers to an organ, tissue, cell, body fluid, or a mixture thereof contained in a living body. Specific examples may include skin, respiratory tract, intestinal tract, urogenital tract, nerve, tumor, bone marrow, blood cells, blood (whole blood, plasma, serum), lymph, cerebrospinal fluid, intraperitoneal fluid, synovial fluid, intrapulmonary fluid, saliva, sputum, urine, and such. Also included in the samples of the present invention are those obtained by washing them or those obtained by culturing them ex vivo. A preferred sample in the present invention is blood, and a particularly preferred sample is plasma or serum.

Alternatively, when the form of the marker is a polynucleotide, whole blood or blood cell fraction can be used as a sample. From these blood-derived samples, the level of expression of a neutrophil-associated gene in the blood can be determined. Methods of extracting mRNA from whole blood and assessing its expression levels are known. In the present invention, comparison of the expression levels of neutrophil-associated genes in blood is a preferred method for marker evaluation. Thus, in a preferred embodiment of the present invention, neutrophil-associated genes can be used as blood markers.

For example, the PAXgene Blood RNA System (QIAGEN) is a combination of blood collection tubes that stabilize blood RNA after blood collection and a general-purpose kit consisting of columns and such for RNA extraction. The expression levels of neutrophil-associated genes in the blood can be readily assessed by utilizing such commercially available tools for RNA analysis.

In the present invention, samples obtained from patients and such may be processed by methods such as enrichment, refining, extraction, isolation, or physical/chemical treatment before measurement of the expression levels of the markers. For example, blood cells or plasma components may be isolated from a blood sample, and DNA or RNA may be extracted from a tissue/cell sample. Alternatively, unwanted components may be denatured/removed with heating or chemical reagents. Such processing is performed mainly for the purpose of improving the stability of the marker, the sensitivity and specificity of measuring its expression level, and such.

Using the expression level of a neutrophil-associated gene as an indicator, the present invention predicts or determines the therapeutic effect of an IL-6 inhibitor for a neutrophil-associated disease. In the present invention, the “IL-6 inhibitor” is not limited as long as the IL-6 inhibitor is capable of blocking IL-6 signal transduction and inhibiting the biological activity of IL-6. Specific examples of the IL-6 inhibitor can include, but are not limited to, a substance that binds to IL-6, a substance that binds to an IL-6 receptor, and a substance that binds to gp130. Other examples of the IL-6 inhibitor can include, but are not limited to, a substance that inhibits phosphorylation of STATS, which is important for the intracellular signaling of IL-6, for example, AG490. The IL-6 inhibitor includes, without being particularly limited, an anti-IL-6 antibody, an anti-IL-6 receptor antibody, an anti-gp130 antibody, an IL-6 variant, a soluble IL-6 receptor variant, a partial IL-6 peptide, a partial IL-6 receptor peptide, and a low-molecular weight compound exhibiting activity similar thereto.

Examples of the preferred embodiment of the IL-6 inhibitor can include an IL-6 receptor inhibitor, particularly an anti-IL-6 receptor antibody.

The origin of the antibody used in the present invention is not particularly limited, and the antibody can be derived from preferably a mammal, more preferably a human

The antibody used in the present invention can be obtained as a polyclonal or monoclonal antibody by use of an approach known in the art. The antibody used in the present invention is particularly preferably a mammal-derived monoclonal antibody. The mammal-derived monoclonal antibody includes an antibody produced by a hybridoma, and an antibody produced by a host transformed with an expression vector containing an antibody gene by a genetic engineering approach. Usually, this antibody blocks the transmission of the biological activity of IL-6 into a cell through its binding to IL-6, an IL-6 receptor, gp130, or the like.

Basically, the monoclonal antibody-producing hybridoma can be prepared by use of a technique known in the art as follows: an IL-6 receptor, IL-6, gp130, or the like is used as a sensitizing antigen in immunization according to an ordinary immunization method, and the resulting immunocytes are fused with parent cells known in the art by an ordinary cell fusion method, and the fused cells are screened for monoclonal antibody-producing cells by an ordinary screening method to prepare monoclonal antibody-producing hybridomas.

Specifically, the monoclonal antibody can be prepared as follows: in the case of preparing, for example, an anti-IL-6 receptor antibody, a human IL-6 receptor or mouse IL-6 receptor to be used as a sensitizing antigen is obtained by using the nucleotide sequence of the IL-6 receptor gene and/or the amino acid sequence of the IL-6 receptor protein disclosed in European Patent Application Publication No. EP 325474 or disclosed in JP-A (Kokai) H3-155795, respectively.

There are two types of IL-6 receptor proteins: a protein expressed on the cell membrane, and a protein dissociated from the cell membrane (soluble IL-6 receptor) (Yasukawa, K. et al., J. Biochem. (1990) 108, 673-676). The soluble IL-6 receptor is constituted by substantially the extracellular region of the IL-6 receptor bound with the cell membrane, and differs from the membrane-bound IL-6 receptor in that the soluble IL-6 receptor lacks the transmembrane region or lacks the transmembrane region and the intracellular region. Any IL-6 receptor may be used as the IL-6 receptor protein as long as the IL-6 receptor may be used as a sensitizing antigen in the preparation of an anti-IL-6 receptor antibody used in the present invention.

The gene sequence of the IL-6 receptor is inserted to an expression vector system known in the art, and appropriate host cells are transformed therewith. Then, the IL-6 receptor protein of interest is purified by a method known in the art from the inside of the host cells or from a culture supernatant thereof. This purified IL-6 receptor protein can be used as the sensitizing antigen. Alternatively, cells expressing the IL-6 receptor or a fusion protein of the IL-6 receptor protein with another protein may be used as the sensitizing antigen.

Likewise, in the case of using IL-6 as a sensitizing antigen in antibody obtainment, human IL-6 is obtained by using the nucleotide sequence of the IL-6 gene and/or the amino acid sequence of the IL-6 protein disclosed in Eur. J. Biochem (1987) 168, 543-550, J. Immunol. (1988)140, 1534-1541, or Agr. Biol. Chem. (1990) 54, 2685-2688. Also, the nucleotide sequence of the gp130 gene and/or the amino acid sequence of the gp130 protein disclosed in EP 411946 can be used as a sensitizing antigen for obtaining an anti-gp130 antibody.

The mammal to be immunized with the sensitizing antigen is not particularly limited and is preferably selected in consideration with compatibility with the parent cells for use in cell fusion. In general, a rodent, for example, a mouse, a rat, or a hamster is used.

The animal is immunized with the sensitizing antigen according to a method known in the art. For example, a general method involves intraperitoneally or subcutaneously injecting the sensitizing antigen to the mammal. Specifically, the sensitizing antigen diluted or suspended in an appropriate volume of phosphate-buffered saline (PBS), saline, or the like is mixed, if desired, with an appropriate amount of a usual adjuvant, for example, a complete Freund's adjuvant. After emulsification, several shots of the emulsion are each preferably administered to the mammal every 4 to 21 days. Also, an appropriate carrier can be used in the immunization with the sensitizing antigen.

After such immunization and confirmation of a rise in desired antibody level in serum, immunocytes are collected from the mammal and subjected to cell fusion. Preferred examples of the immunocytes that are subjected to cell fusion particularly include spleen cells.

Mammalian myeloma cells for use as partner parent cells to be fused with the immunocytes have already been known in the art, and various cell lines, for example, P3X63Ag8.653 (Kearney, J. F. et al., J. Immunol. (1979) 123, 1548-1550), P3X63Ag8U.1 (Current Topics in Microbiology and Immunology (1978) 81, 1-7), NS-1 (Kohler. G. and Milstein, C. Eur. J. Immunol. (1976) 6, 511-519), MPC-11 (Margulies. D.H. et al., Cell (1976) 8, 405-415), SP2/0 (Shulman, M. et al., Nature (1978) 276, 269-270), FO (de St. Groth, S. F. et al., J. Immunol. Methods (1980) 35, 1-21), 5194 (Trowbridge, I. S. J. Exp. Med. (1978) 148, 313-323), and R210 (Galfre, G. et al., Nature (1979) 277, 131-133) are appropriately used.

Basically, the cell fusion between the immunocytes and the myeloma cells can be carried out according to a method known in the art, for example, the method of Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46).

More specifically, the cell fusion is carried out, for example, in a usual nutrient medium in the presence of a cell fusion promoter. For example, polyethylene glycol (PEG) or hemagglutinating virus of Japan (HVJ) is used as the fusion promoter. An auxiliary such as dimethyl sulfoxide can be further added thereto and used, if desired, for enhancing fusion efficiency.

The ratio between the immunocytes and the myeloma cells used is preferably set to, for example, 1:1 to 10:1 (immunocytes:myeloma cells). For example, an RPMI1640 medium or a MEM medium suitable for the growth of the myeloma cell lines mentioned above or a usual medium for use in this kind of cell culture can be used in the cell fusion and may be used in combination with a serum supplement such as fetal calf serum (FCS).

For the cell fusion, predetermined amounts of the immunocytes and the myeloma cells are well mixed in the medium. A PEG solution, for example, a solution of PEG having an average molecular weight of about 1000 to 6000, preheated to approximately 37° C. is usually added to the mixture at a concentration of 30 to 60% (w/v) and mixed therewith to form the fusion cells (hybridomas) of interest. Subsequently, the cell fusion agent and the like unfavorable for the growth of the hybridomas can be removed by repeating the operation of sequentially adding an appropriate medium and removing a supernatant by centrifugation.

The hybridomas thus obtained are cultured in a usual selective medium, for example, a HAT medium (medium containing hypoxanthine, aminopterin, and thymidine) for selection. This culture in the HAT medium is continued for a period (usually, several days to several weeks) sufficient for killing cells (non-fused cells) other than the hybridomas of interest. Subsequently, hybridomas producing the antibody of interest are screened for and cloned by an ordinary limiting dilution method.

In addition to obtaining such hybridomas by immunizing a non-human animal with an antigen, a desired human antibody having binding activity against a desired antigen or against cells expressing the antigen may be obtained by sensitizing in vitro human lymphocytes with the desired antigen protein or cells expressing the antigen and fusing the sensitized B lymphocytes with human myeloma cells, for example, with U266 (see JP-A (Kokai) H1-59878). Alternatively, the antigen or cells expressing the antigen may be administered to a transgenic animal having a human antibody gene repertoire, and the desired human antibody can be obtained according to the method mentioned above (see International Patent Application Publication Nos. WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, and WO96/33735).

The monoclonal antibody-producing hybridomas thus prepared can be passaged in a usual medium and can also be preserved for a long period in liquid nitrogen.

The monoclonal antibody is obtained from the hybridomas by employing, for example, a method which involves culturing the hybridomas according to an ordinary method and obtaining the antibody as a culture supernatant thereof, or a method which involves administering the hybridomas to mammals compatible therewith and, after growth, obtaining the antibody as ascitic fluid thereof. The former method is suitable for obtaining a highly pure antibody, while the latter method is suitable for the large-scale production of the antibody.

For example, hybridomas producing an anti-IL-6 receptor antibody can be prepared by a method disclosed in JP-A (Kokai) H3-139293. This preparation can be carried out by a method which involves intraperitoneally injecting PM-1 antibody-producing hybridomas to BALB/c mice to obtain ascitic fluid, and purifying the PM-1 antibody from the ascitic fluid, or a method which involves culturing the hybridomas in an appropriate medium, for example, an RPMI1640 medium containing 10% fetal calf serum and 5% BM-Condimed H1 (manufactured by Boehringer Mannheim), a Hybridoma SFM medium (manufactured by Gibco BRL/Life Technologies, Inc.), or a PFHM-II medium (manufactured by Gibco BRL/Life Technologies, Inc.) and purifying the PM-1 antibody from the culture supernatant.

In the present invention, a recombinant antibody produced by use of a gene recombination technique which involves cloning an antibody gene from hybridomas, incorporating the antibody gene into an appropriate vector, and transferring this vector to a host can be used as the monoclonal antibody (see e.g., Borrebaeck C.A.K. and Larrick J. W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

Specifically, mRNAs encoding the variable (V) regions of the antibody are isolated from cells, for example, hybridomas, producing the antibody of interest. For the mRNA isolation, total RNA is prepared by a method known in the art, for example, a guanidine ultracentrifugation method (Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or an AGPC method (Chomczynski, P. et al., Anal. Biochem. (1987) 162, 156-159), and the mRNAs are prepared using mRNA Purification Kit (manufactured by Pharmacia Corp.) or the like. Alternatively, the mRNAs can be directly prepared by use of QuickPrep mRNA Purification Kit (manufactured by Pharmacia Corp.).

Antibody V region cDNAs are synthesized from the obtained mRNAs using reverse transcriptase. The cDNA synthesis can be carried out using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit or the like. Also, 5′-Ampli FINDER RACE Kit (manufactured by Clontech Laboratories, Inc.) and a PCR-based 5′-RACE method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; and Belyaysky, A.et al., Nucleic Acids Res. (1989) 17, 2919-2932) can be used in the cDNA synthesis and amplification. The DNA fragments of interest are purified from the obtained PCR products and ligated with vector DNAs. Recombinant vectors are thereby prepared and transferred to E. coli or the like. Colonies are selected, and desired recombinant vectors are prepared. The nucleotide sequences of the DNAs of interest are confirmed by a method known in the art, for example, a deoxy method.

If DNAs encoding the V regions of the antibody of interest are obtained, these DNAs are linked to DNAs encoding constant regions (C regions) of a desired antibody, and these linked DNAs are incorporated into expression vectors. Alternatively, the DNAs encoding the antibody V regions may be incorporated into expression vectors containing the DNAs of the antibody C regions.

For the production of the antibody used in the present invention, the antibody gene is incorporated into an expression vector such that the antibody gene is expressed under the control of expression control regions, for example, an enhancer and a promoter, as mentioned later. Next, host cells are transformed with this expression vector, and the antibody can be expressed.

In the present invention, a recombinant antibody that has been artificially engineered for the purpose of, for example, reducing the heterologous antigenicity against humans, for example, a chimeric antibody or a humanized antibody, can be used. Such an engineered antibody can be produced by use of a known method.

A chimeric antibody is obtained by linking the antibody V region-encoding DNAs obtained as described above to human antibody C region-encoding DNAs, and incorporating the linked DNAs into expression vectors, which are then introduced into a host, followed by production of the antibody (see EP125023 and WO92-19759). A chimeric antibody useful in the present invention can be obtained by use of this known method.

The humanized antibody is also called reshaped human antibody or antibody made into human type antibody, and is obtained by grafting the complementarity-determining regions (CDRs) of a non-human mammalian antibody, for example, a mouse antibody, to the complementarity-determining regions of a human antibody. A general gene recombination approach therefor is also known (see EP125023 and WO92-19759).

Specifically, DNA sequences designed so as to link mouse antibody CDRs and human antibody framework regions (FRs) are synthesized by PCR using several prepared oligonucleotides having overlapping terminal portions. The obtained DNAs are linked to DNAs encoding human antibody C regions. Subsequently, the linked DNAs are incorporated into expression vectors, which are then transferred to a host, followed by the production of the antibody to obtain the humanized antibody (see EP239400 and WO92-19759).

The human antibody FRs to be connected via CDRs are selected such that the complementarity-determining regions form a favorable antigen-binding site. If necessary, amino acids in the framework regions of the antibody variable regions may be substituted such that the complementarity-determining regions of the resulting reshaped human antibody form an appropriate antigen-binding site (Sato, K. et al., Cancer Res. (1993) 53, 851-856).

Usually, human antibody C regions are used for the chimeric antibody or the humanized antibody. Examples of the human antibody heavy chain C region include Cy. For example, Cy1, Cy2, Cy3, or Cy4 can be used. Examples of the human antibody light chain C region can include x and X. These human antibody C regions may be modified in order to improve the stability of the antibody or the stability of production thereof.

A chimeric antibody is composed of the variable regions of a non-human mammal-derived antibody and human antibody-derived C regions. A humanized antibody is composed of the complementarity-determining regions of a non-human mammal-derived antibody and human antibody-derived framework regions and C regions. These antibodies exhibit reduced antigenicity in human bodies and as such, are useful as antibodies for use as pharmaceuticals.

Preferable specific examples of the humanized antibody used in the present invention include humanized PM-1 antibodies (see WO92-19759).

In addition to the aforementioned methods for obtaining a human antibody, a technique of obtaining a human antibody by panning using a human antibody library is also known. For example, human antibody variable regions are expressed as a single-chain antibody (scFv) on the surface of phages by a phage display method, and a phage binding to the antigen may be selected. The gene of the selected phage can be analyzed to determine DNA sequences encoding the variable regions of the human antibody binding to the antigen. If the DNA sequence of scFv binding to the antigen is revealed, an appropriate expression vector containing this sequence can be prepared to obtain the human antibody. These methods have already been well known. See WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388.

The antibody gene constructed as described above can be expressed by a method known in the art. In the case of using mammalian cells, the antibody gene can be expressed by use of a DNA in which a routinely used useful promoter, the antibody gene to be expressed, and a poly-A signal 3′-downstream thereof are functionally linked, or by use of a vector containing the DNA. Examples of the promoter/enhancer can include human cytomegalovirus immediate early promoter/enhancer.

Alternatively, a promoter/enhancer of a virus such as retrovirus, polyoma virus, adenovirus, or simian virus 40 (SV40), a mammalian cell-derived promoter/enhancer such as human elongation factor 1α (HEF1α), or the like can be used as the promoter/enhancer for the antibody expression used in the present invention.

In the case of using, for example, the SV40 promoter/enhancer, the antibody expression can be readily carried out according to the method of Mulligan et al. (Mulligan, R. C. et al., Nature (1979) 277, 108-114). In the case of using the HEF1α promoter/enhancer, the antibody expression can be readily carried out according to the method of Mizushima et al. (Mizushima, S and Nagata, S. Nucleic Acids Res. (1990) 18, 5322).

In the case of using prokaryotic cells as the host, bacterial cells can be used in the production system. E. coli and Bacillus subtilis are known as the bacterial cells.

For E. Coli, a routinely used useful promoter, a signal sequence for antibody secretion, and the antibody gene to be expressed can be functionally linked and expressed. Examples of the promoter can include lacZ promoter and araB promoter. In the case of using the lacZ promoter, the method of Ward et al. (Ward, E. S. et al., Nature (1989) 341, 544-546; and Ward, E. S. et al. FASEB J. (1992) 6, 2422-2427) can be followed. In the case of using the araB promoter, the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043) can be followed.

In the case of production in the periplasm of E. coli, pelB signal sequence (Lei, S. P. et al., J. Bacteriol. (1987) 169, 4379-4383) can be used as the signal sequence for antibody secretion. The antibodies produced in the periplasm are separated and then used after appropriate refolding of the antibody structure (see e.g., WO96/30394).

A replication origin derived from SV40, polyoma virus, adenovirus, bovine papillomavirus (BPV), or the like can be used. The expression vector can contain a selective marker such as aminoglycoside phosphotransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, or dihydrofolate reductase (dhfr) gene in order to amplify the number of gene copies in the host cell system.

For the production of the antibody used in the present invention, an arbitrary production system can be used. There are in vitro and in vivo production systems for antibody production. Examples of the in vitro production system include a production system using eukaryotic cells and a production system using prokaryotic cells.

In the case of using eukaryotic cells as the host, animal cells, plant cells, or fungal cells can be used in the production system. (1) Mammalian cells, for example, CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, and Vero, (2) amphibian cells, for example, Xenopus oocytes, or (3) insect cells, for example, sf9, sf21, and Tn5 are known as the animal cells. Nicotiana tabacum-derived cells are known as the plant cells and can be callus-cultured. Yeasts of, for example, the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or filamentous fungi of, for example, the genus Aspergillus (e.g., Aspergillus niger) are known as the fungal cells.

The antibody gene of interest is transferred to these cells by transformation, and the transformed cells are cultured in vitro to obtain the antibody. This culture is carried out according to a method known in the art. For example, DMEM, MEM, RPMI1640, or IMDM can be used as a medium and may be used in combination with a serum supplement such as fetal calf serum (FCS). Alternatively, the cells thus harboring the antibody gene may be transferred to the peritoneal cavity or the like of an animal so that the antibody is produced in vivo.

On the other hand, examples of the in vivo production system include a production system using an animal and a production system using a plant. When an animal is used, a mammal, an insect, or the like can be used in the production system.

A goat, a pig, sheep, a mouse, cattle, or the like can be used as the mammal (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). A silkworm can be used as the insect. In the case of using the plant, for example, tobacco can be used.

The antibody gene is introduced into such an animal or a plant, and the antibody is produced in the body of the animal or the plant and recovered. For example, the antibody gene is prepared as a fusion gene by inserting the gene midway into a gene encoding a protein specifically produced in milk, such as goat 13 casein. A DNA fragment that contains the fusion gene having the inserted antibody gene is injected into a goat embryo, and this embryo is introduced into a female goat. The desired antibody is obtained from milk produced by a transgenic goat born from the embryo-recipient goat, or progeny thereof. Hormones may be appropriately used for the transgenic goat in order to increase the amount of the milk containing the desired antibody produced by the transgenic goat (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).

In the case of using a silkworm, a silkworm is infected with baculovirus having an insert of the antibody gene of interest, and the desired antibody is obtained from the body fluid of this silkworm (Maeda, S. et al., Nature (1985) 315, 592-594). In the case of using tobacco, the antibody gene of interest is inserted to a vector for expression in plants, for example, pMON530, and this vector is introduced into a bacterium such as Agrobacterium tumefaciens. Tobacco, for example, Nicotiana tabacum, is infected with this bacterium, and the desired antibody is obtained from the leaf of this tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. (1994)24, 131-138).

In the case of producing an antibody in the in vitro or in vivo production system as mentioned above, an antibody heavy chain (H chain)-encoding DNA and an antibody light chain (L chain)-encoding DNA may be incorporated into separate expression vectors, and the host can be co-transformed with these expression vectors. Alternatively, the H chain-encoding DNA and the L chain-encoding DNA may be incorporated into a single expression vector, and the host can be transformed with this expression vector (see WO94-11523).

The antibody used in the present invention may be a fragment of the antibody or a modified form of the antibody as long as the fragment or the modified form can be suitably used in the present invention. Examples of the antibody fragment include Fab, F(ab′)2, Fv, and single-chain Fv (scFv) containing H and L chain Fvs linked through an appropriate linker.

Specifically, the antibody fragment is formed by the treatment of the antibody with an enzyme, for example, papain or pepsin, or is expressed in appropriate host cells after construction of a gene encoding the antibody fragment and subsequent transfer of this gene to an expression vector (see e.g., Co, M.S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. & Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496; Plueckthun, A. & Skerra, A. Methods in Enzymology (1989) 178, 497-515; Lamoyi, E., Methods in Enzymology (1989) 121, 652-663; Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-66; and Bird, R. E. et al., TIBTECH (1991) 9, 132-137).

An scFv is obtained by linking the H chain V region and the L chain V region of an antibody. In this scFv, the H chain V region and the L chain V region are linked via a linker, preferably a peptide linker (Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883). The H chain V region and the L chain V region in the scFv may be derived from any of the above-described antibodies according to the present invention. For example, an arbitrary single-chain peptide composed of 12 to 19 amino acid residues is used as the peptide linker for linking the V regions.

A DNA encoding the scFv is obtained by using a DNA encoding the H chain or the H chain V region of the aforementioned antibody and a DNA encoding the L chain or the L chain V region of the antibody as templates, and amplifying from each of those sequences a DNA portion encoding the desired amino acid sequence by PCR using a primer pair defining the two ends thereof, followed by further amplification using a DNA encoding the peptide linker portion and a primer pair that is designed such that each of the two ends of the peptide linker is linked to the H chain and the L chain, respectively.

Once the scFv-encoding DNA is prepared, an expression vector containing the DNA, and a host transformed with the expression vector can be obtained according to routine methods. Also, the scFv can be obtained according to a routine method using the host.

These antibody fragments can be produced through obtainment and expression of their genes and production by the host in the same way as above. The “antibody” according to the present invention also encompasses these antibody fragments.

Antibodies bound with various molecules such as polyethylene glycol (PEG) may be used as modified forms of antibody. The “antibody” according to the present invention also encompasses these modified forms of the antibody. Such a modified form of the antibody can be obtained by chemical modification of the obtained antibody. These methods have already been established in the art.

The antibody produced and expressed as described above can be separated from the inside or outside of the cells or from the host and purified to homogeneity. The separation and purification of the antibody used in the present invention can be carried out by affinity chromatography. Examples of columns for use in the affinity chromatography include protein A columns and protein G columns Examples of carriers for use in the protein A columns include Hyper D, POROS, and Sepharose F. F. Any of other ordinary separation and purification methods for use in proteins can be used without limitation.

For example, the antibody used in the present invention can be separated and purified by appropriately selecting or combining chromatography other than the affinity chromatography, filters, ultrafiltration, salting out, and/or dialysis. Examples of the chromatography include ion-exchange chromatography, hydrophobic chromatography, and gel filtration. These chromatography techniques are applicable to high-performance liquid chromatography (HPLC). Alternatively, reverse-phase HPLC may be used.

The concentration of the antibody thus obtained can be measured by, for example, absorbance measurement or ELISA. Specifically, in the case of measuring the concentration by the absorbance measurement, the absorbance is measured at 280 nm after appropriate dilution of the antibody with PBS(-), and the concentration is calculated assuming that 1 mg/ml is 1.35 OD. Alternatively, the concentration can be measured by ELISA as follows: 100 μl of goat anti-human IgG (manufactured by TAG) diluted to 1 μg/ml with a 0.1 M bicarbonate buffer solution (pH 9.6) is added to a 96-well plate (manufactured by Nunc/Thermo Fisher Scientific, Inc.) and incubated overnight at 4° C. to immobilize the antibody thereon. After blocking, 100 μl of an appropriately diluted antibody used in the present invention or a sample containing the antibody, or a preparation human IgG (manufactured by Cappel Laboratories, Inc.) is added thereto and incubated at room temperature for 1 hour.

After washing, 100 μl of alkaline phosphatase-labeled anti-human IgG (manufactured by BioSource International, Inc.) diluted 5000-fold is added thereto and incubated at room temperature for 1 hour. After washing, a substrate solution is added thereto and incubated. Then, the absorbance is measured at 405 nm using MICROPLATE READER Model 3550 (manufactured by Bio-Rad Laboratories, Inc.) to calculate the concentration of the antibody of interest.

Specific examples of the anti-IL-6 antibody can include, but are not particularly limited to, MH166 (Matsuda, T. et al., Eur. J. Immunol. (1998) 18, 951-956) and SK2 antibody (Sato K et al., Academic proceedings of the 21st General Meeting of the Japanese Society for Immunology (1991) 21, 166).

Specific examples of the anti-IL-6 receptor antibody include, but are not particularly limited to, MR16-1 antibody (Tamura, T. et al. Proc. Natl. Acad. Sci. USA (1993) 90, 11924-11928), PM-1 antibody (Hirata, Y. et al., J. Immunol. (1989) 143, 2900-2906), AUK12-20 antibody, AUK64-7 antibody, and AUK146-15 antibody (WO92-19759). Among them, preferred examples of the monoclonal antibody against the human IL-6 receptor include, but are not limited to, the PM-1 antibody, and preferred examples of the monoclonal antibody against the mouse IL-6 receptor include, but are not limited to, the MR16-1 antibody. Preferred examples of the humanized anti-IL-6 receptor antibody can include, but are not limited to, an antibody comprising a heavy chain variable region having the sequence of SEQ ID NO: 19 and a light chain variable region having the sequence of SEQ ID NO: 20, an antibody comprising a heavy chain having the sequence of SEQ ID NO: 21 and a light chain having the sequence of SEQ ID NO: 22, a humanized PM-1 antibody (Tocilizumab, MRA), and SA237. Other preferred examples of the humanized anti-IL-6 receptor antibody can include, but are not limited to, antibodies described in WO2009/041621 and WO2010/035769. Examples of other preferred embodiments of the anti-IL-6 receptor antibody can include, but are not limited to, an anti-IL-6 receptor antibody that recognizes the same epitope as that recognized by the humanized PM-1 antibody (Tocilizumab, MRA) and an anti-IL-6 receptor antibody that recognizes the same epitope as that recognized by SA237.

Specific examples of the anti-gp130 antibody include, but are not particularly limited to, AM64 antibody (JP-A (Kokai) H3-219894), 4B11 antibody, 2H4 antibody (U.S. Pat. No. 5,571,513), and B-P8 antibody (JP-A (Kokai) H8-291199).

The IL-6 variant used in the present invention is a substance that has binding activity with the IL-6 receptor and does not transduce the biological activity of IL-6. Thus, the IL-6 variant blocks the signal transduction of IL-6 because it competes with IL-6 for binding to the IL-6 receptor but does not transduce the biological activity of IL-6.

An IL-6 variant is prepared by introducing mutations into IL-6 through substitution of amino acid residues in the amino acid sequence of IL-6. The origin of IL-6 on which the IL-6 variant is based is not limited and is preferably human IL-6 in consideration of antigenicity, etc. Specifically, the secondary structure of the amino acid sequence of IL-6 is predicted by use of a molecular modeling program known in the art, for example, WHATIF (Vriend et al., J. Mol. Graphics (1990) 8, 52-56), and the influence of amino acid residues to be substituted on the whole structure is evaluated. After determination of appropriate amino acid residues to be substituted, a vector containing a nucleotide sequence encoding the human IL-6 gene is used as a template, and a conventional PCR method is performed to introduce mutations such that the amino acids are substituted, and thereby a gene encoding the IL-6 variant is obtained. This gene is incorporated into an appropriate expression vector according to the need, and the IL-6 variant can be obtained according to the aforementioned expression, production, and purification methods for the recombinant antibody.

Specific examples of the IL-6 variant can include IL-6 variants disclosed in Brakenhoff et al., J. Biol. Chem. (1994) 269, 86-93, Savino et al., EMBO J. (1994) 13, 1357-1367, WO96-18648, and WO96-17869.

A partial IL-6 receptor peptide is a peptide having a portion or the whole of the amino acid sequence of a region involved in the binding of the IL-6 receptor to IL-6 in the amino acid sequence of the IL-6 receptor. Such a peptide is composed of usually 10 to 80, preferably 20 to 50, more preferably 20 to 40 amino acid residues.

The partial IL-6 receptor peptide can be prepared by identifying the region involved in the binding of the IL-6 receptor to IL-6 in the amino acid sequence of the IL-6 receptor and producing the peptide by a conventionally known method, for example, a genetic engineering approach or a peptide synthesis method on the basis of a portion or the whole of the amino acid sequence of the identified region.

For the preparation of the partial IL-6 receptor peptide by the genetic engineering approach, a DNA sequence encoding the desired peptide is incorporated into an expression vector, and the partial IL-6 receptor peptide can be obtained according to the aforementioned expression, production, and purification methods for the recombinant antibody.

For the preparation of the partial IL-6 receptor peptide by the peptide synthesis method, a method conventionally used in peptide synthesis, for example, a solid-phase synthesis method or a liquid-phase synthesis method can be used.

Specifically, the peptide synthesis can be carried out according to methods described in Zoku Iyakuhin no Kaihatsu (Development of Pharmaceuticals, Second Series, in English), Vol. 14, Peptide Synthesis, edited by Haruaki Yajima, Hirokawa Shoten Co., Ltd. (1991). The solid-phase synthesis method used is a method which involves, for example, coupling an amino acid corresponding to the C terminus of a peptide to be synthesized to a support insoluble in an organic solvent, and elongating a peptide chain by alternately repeating the reaction of condensing one amino acid at a time (its α-amino group and side chain functional groups have been protected with appropriate protective groups) in a direction from the C terminus toward the N terminus and the reaction of eliminating the protective group of the α-amino group of the amino acid or peptide bound onto the resin. The solid-phase peptide synthesis method is broadly divided into Boc and Fmoc methods depending on the types of the protective groups used.

After such synthesis of the peptide of interest, deprotection reaction and cleavage reaction of the peptide chain from the support are carried out. In the cleavage reaction of the peptide chain, usually, hydrogen fluoride or trifluoromethanesulfonic acid can be used for the Boc method, and TFA can be used for the Fmoc method. In the Boc method, the protected peptide resin is treated, for example, in the presence of anisole in hydrogen fluoride. Subsequently, protective group elimination and cleavage from the support are carried out to recover the peptide. This peptide is freeze-dried to obtain a crude peptide. On the other hand, in the Fmoc method, for example, deprotection reaction and cleavage reaction of the peptide chain from the support can be carried out by the same operation as above in TFA.

The obtained crude peptide can be separated and purified by application to HPLC. The peptide can be eluted under the optimum conditions by use of a water-acetonitrile mixed solvent conventionally used in protein purification. A fraction corresponding to a peak in the obtained profile of chromatography is separated and then freeze-dried. The peptide fraction thus purified is identified by, for example, mass spectrometric molecular weight analysis, amino acid composition analysis, or amino acid sequence analysis.

The present invention also relates to an agent for treating neutrophil-associated diseases with high expression level of a neutrophil-associated gene comprising an IL-6 inhibitor as an active ingredient. The “neutrophil-associated diseases with high expression level of a neutrophil-associated gene” herein preferably refers to neutrophil-associated diseases in which the expression level of a neutrophil-associated gene is high prior to administration of an IL-6 inhibitor, but the expression level of a neutrophil-associated gene is reduced following administration compared to before administration of the IL-6 inhibitor.

In the present invention, the phrase “comprising as an active ingredient” means comprising an IL-6 inhibitor as at least one of the active ingredients and does not limit the content percentage thereof. The therapeutic agent of the present invention may also contain active ingredients other than the IL-6 inhibitor. The therapeutic agent of the present invention may be used not only for therapeutic purposes but for preventive purposes.

The therapeutic agent of the present invention can be formulated according to a routine method (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A). The therapeutic agent of the present invention may contain a pharmaceutically acceptable carrier and/or additive according to needs. The therapeutic agent of the present invention can contain, for example, a surfactant (PEG, Tween, etc.), an excipient, an antioxidant (ascorbic acid, etc.), a colorant, a flavoring agent, a preservative, a stabilizer, a buffer (phosphate, citrate, other organic acids, etc.), a chelating agent (EDTA, etc.), a suspending agent, a tonicity agent, a binder, a disintegrant, a lubricant, a flowability enhancer, and a corrigent. However, the therapeutic agent of the present invention may appropriately contain, without being limited to the above agents, other carriers routinely used. Specific examples thereof can include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, white soft sugar, carboxymethylcellulose, corn starch, and inorganic salts. Also, the therapeutic agent of the present invention may contain other low-molecular-weight polypeptides, proteins (e.g., serum albumin, gelatin, and immunoglobulin), and amino acids. In the case of preparing an aqueous solution for injection, the IL-6 inhibitor is dissolved in, for example, an isotonic solution containing saline, glucose, or other adjuvants. Examples of the adjuvants include D-sorbitol, D-mannose, D-mannitol, and sodium chloride. The solution may be further used in combination with an appropriate solubilizer, for example, an alcohol (ethanol, etc.), a polyalcohol (propylene glycol, PEG, etc.), or a nonionic surfactant (polysorbate 80 or HCO-50).

The IL-6 inhibitor may be enclosed in a microcapsule (microcapsule made of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], or the like) or prepared into a colloid drug delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (see e.g., Remington's Pharmaceutical Science 16th edition & Oslo Ed. (1980)). Methods for formulating drugs as sustained-release drugs are also known in the art and may be applied to the IL-6 inhibitor of the present invention (Langer et al., J. Biomed. Mater. Res. (1981) 15: 167-277; Langer, Chem. Tech. (1982) 12: 98-105; U.S. Pat. No. 3,773,919; EP 58,481; Sidman et al., Biopolymers (1983) 22: 547-56; and EP 133,988). In addition, the therapeutic agent of the present invention may be supplemented or mixed with hyaluronidase to allow an increased amount of fluid to be subcutaneously administered (e.g., WO2004/078140).

The therapeutic agent of the present invention can be administered through any of oral and parenteral routes and is preferably administered parenterally. Specifically, the therapeutic agent of the present invention is administered to a patient through injection or percutaneous administration. Examples of the dosage form of the injection include systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or such. The therapeutic agent of the present invention may be injected locally, particularly, intramuscularly, to a treatment site or the neighborhood thereof. Examples of the dosage form of the percutaneous administration include ointments, gels, creams, poultices, and patches, which permit systemic or local administration. The administration method can be appropriately selected according to the age and symptoms of a patient. The dose can be selected, for example, within the range of 0.0001 mg to 100 mg of the active ingredient per kg of body weight per dose. Alternatively, for example, when administering to a human patient, the range of 0.001 to 1000 mg/kg body weight of the active ingredient per patient can be selected. The single dose preferably contains, for example, approximately 0.01 to 50 mg/kg body weight of the antibody of the present invention. However, the therapeutic agent of the present invention is not limited by these doses.

The therapeutic agent of the present invention can be used alone for treating neutrophil-associated diseases with high expression levels of neutrophil-associated genes in humans and animals. Alternatively, the therapeutic agent of the present invention can be administered orally as a mixture with other ingredients that may be commonly used in pharmaceuticals and foods. The therapeutic agent of the present invention can also be used in combination with other compounds, microbes, or the like known to have therapeutic and/or preventive effect on neutrophil-associated diseases.

The present invention also relates to a method for treating neutrophil-associated diseases in a subject having high levels of expression of neutrophil-associated genes, which comprises administering an IL-6 inhibitor to the subject. Subjects to which an IL-6 inhibitor is administered include mammals Mammals include humans and non-human mammals in need of treatment or prevention of a neutrophil-associated disease, preferably humans and monkeys, and more preferably humans.

The present invention also relates to an IL-6 inhibitor for use in the treatment of neutrophil-associated diseases with high expression levels of neutrophil-associated genes. Alternatively, the present invention relates to the use of an IL-6 inhibitor in the manufacture of an agent for treating neutrophil-associated diseases with high expression levels of neutrophil-associated genes.

The present invention also relates to a method for manufacturing an agent for treating neutrophil-associated diseases with high expression levels of neutrophil-associated genes, comprising mixing an IL-6 inhibitor with a pharmaceutically acceptable carrier. All prior art documents cited herein are incorporated herein by reference.

EXAMPLES

Next, the present invention will be described more specifically with examples; however, the present invention is not limited to the examples below.

As described below, the present inventors analyzed by DNA microarray, genes that normalized in neuromyelitis optica (NMO) patients after administration of tocilizumab (TCZ).

Gene expression assay

Among NMO patients who received TCZ by intravenous injection once every four weeks at 8 mg/kg each time, seven patients with improved NMO were subject to collection of peripheral blood into the PAXgene blood RNA tubes (PreAnalytiX GmBH) prior to and twelve months after the administration of the first TCZ treatment. Peripheral blood was collected during the day. From the peripheral blood, mRNA was extracted using the PAXgene Blood miRNA spin-column (QIAGEN). From the mRNA, the cDNA was synthesized by One-Color Microarray-Based Gene Expression Analysis (Agilent Technologies), ver. 6.5. 600 ng of cDNA was hybridized with the SurePrint G3 Human GE microarray (Agilent Technologies), ver. 2.0 for 17 hours, washed, and scanned using the Aglient DNA microarray scanner. Scanned image data were quantified using Feature Extraction, ver. 10.7.1.1 software (Agilent Technologies). Data were normalized using the GeneSpring GX 12.0 software (Agilent Technologies). Changes in the expression ratio were analyzed by paired t-test. The expression ratios and variations in gene probes were extracted using the Excel 2013 software. Probes meeting one of the following two criteria were extracted: (1) expression ratio greater than or equal to 1.5 or less than or equal to 0.67, p<0.05; and (2) expression ratio greater than 1 and less than 1.5, or greater than 0.67 and less than 1, p<0.01. Extracted probes were analyzed online using DAVID 6.8 (Reference: Huang DW, Sherman BT, and Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc.4; 44-57 (2009)).

Improvements in NMO were determined based on a decrease in the number of relapses per year, a decrease in the subjective symptoms of pain and fatigue, and a decrease in the number of EDSS (Expanded Disability Status Scale).

Results

The results of the above analysis are shown in FIG. 1 . Gene expressions in the NMO patients and non-NMO patients (controls) were analyzed by DNA microarray using a total number of 50599 probes, showing upregulation of 2219 probes and downregulation of 1890 probes in NMO patients compared to the controls. Also, 71 probes were upregulated and 224 probes were downregulated in patients after TCZ treatment compared with before TCZ treatment. Among the 2175 probes having high expression levels in NMO patients before TCZ treatment, 41 were downregulated after TCZ treatment.

Among the probes that were significantly different from the controls, examples of probes that were downregulated in NMO patients one year after TCZ treatment are shown in Table 1. Table 1 shows probes with a ratio of expression before and after TCZ treatment of less than 0.5, starting from those with greatly reduced expression ratios. Surprisingly, top nine of these genes are those associated with the granule content of neutrophils. These neutrophil-associated genes may be useful as markers for predicting or determining the therapeutic effect of IL-6 inhibitors in neutrophil-associated diseases, since they are genes with significantly higher expression levels in NMO patients and greatly reduced expression levels after TCZ-treatment.

Besides IL-6, neutrophils have been reported to be associated with the disease state of neuromyelitis optica (Non-Patent Documents 3 and 4). In addition to neuromyelitis optica, Neuro-Sweet disease, stroke, cerebral infarction and such are known as diseases in which IL-6 and neutrophil are associated with their pathological conditions (Clinical Neurology 2012; 52:1234-1236). It is believed that for these diseases also, the therapeutic effect of IL-6 inhibitors can be predicted or determined by measuring the expression levels of neutrophil-associated genes in patients.

TABLE 1 Expression ratio Expression Probe Gene (NMO post/ ratio (NMO Name Symbol Gene Name Description NMO pre) pre/control) A_23_P140384 CTSG cathepsin G Homo sapiens 0.102 11.566 cathepsin G (CTSG), mRNA [NM_001911] A_23_P326080 DEFA4 defensin, alpha 4, Homo sapiens 0.139 14.410 corticostatin defensin, alpha 4, corticostatin (DEFA4), mRNA [NM_001925] A_33_P3279353 AZU1 azurocidin 1 Homo sapiens 0.143 9.705 azurocidin 1 (AZU1), mRNA [NM_001700] A_23_P131785 BPI bactericidal/ Homo sapiens 0.144 5.567 permeability- bactencidal/permeability- increasing increasing protein protein (BPI), mRNA [NM_001725] A_23_P130961 ELANE elastase, Homo sapiens 0.147 12.664 neutrophil elastase, neutrophil expressed expressed (ELANE), mRNA [NM_001972] A_23_P166848 LTF lactotransferrin Homo sapiens 0.223 5.936 lactotransferrin (LTF), transcript variant 1. mRNA [NM_002343] A_23_P31816 DEFA3 defensin, alpha 3, Homo sapiens 0.276 7.789 neutrophil- defensin, alpha 3, specific neutrophil-specific (DEFA3), mRNA [NM_005217] A_23_P169437 LCN2 lipocalin 2 Homo sapiens 0.277 5.934 lipocalin 2 (LCN2), mRNA [NM_005564] A_23_P253791 CAMP cathelicidin Homo sapiens 0.319 9.094 antimicrobial cathelicidin peptide antimicrobial peptide (CAMP), mRNA NM_004345] PCR-Analysis of Blood cDNA

TCZ was administered by intravenous injection at 8 mg/kg once every four weeks; and for seven patients with improved NMO, whole blood was collected into the PAXgene RNA blood collection tubes (PreAnalytiX GmBH) and the whole blood mRNA was extracted on the PAXgene Blood miRNA Spin Column (QIAGEN). The PrimeScript RT-PCR Kit (TaKaRa) was used to synthesize cDNA from the extracted mRNA. The SYBER Ex Taq assay was used for RT-PCR, and reactions were monitored with the LightCycler 96 system (Roche). Commercial sets (Quantitect Primer Assay, AIAGEN) were used for the respective primers for amplification below with (3-actin (ACTB) as a control:

Hs_CTSG_1_SG QuantiTect Primer Assay (200) QT00051667 QIAGEN Hs_LTF_2_SG QuantiTect Primer Assay (200) QT01677879 QIAGEN Hs_AZU1_1_SG QuantiTect Primer Assay (200) QT00012327 QIAGEN Hs_BPI_1_SG QuantiTect Primer Assay (200) QT00096649 QIAGEN Hs_DEFA4_1_SG Quanti Tect Primer Assay (200) QT00001603 QIAGEN ß-actin Forward (SEQ ID NO: 23) CACTCTTCCAGCCTTCCTTCC Reverse (SEQ ID NO: 24) GTCTTTGCGGATG

Whole blood was collected before and one year after TCZ administration. Improvements in NMO were determined based on a decrease in the number of relapses per year, a decrease in the subjective symptoms of pain and fatigue, and a decrease in the number of EDSS (Expanded Disability Status Scale).

Statistical Analysis

Wilcoxon's signed rank sum test was carried out to compare the NMO group data before and after TCZ-treatment using the Prism software. The Mann-Whitney test was used to compare the NMO and control groups. A p value less than 0.05 was considered to be significantly different.

Some of the microarray-based analysis results verified by RT-PCR of the whole blood cDNA are shown in FIG. 2 . The top three genes (CTSG, DEFA4, and AZU1) for which significant decreases in the expression levels were observed in the microarray analysis of NMO patients one year after TCZ treatment had a similarly decreasing trend in the expression level by RT-PCR analysis, and this supports the microarray analysis results. On the other hand, such changes in the expression level were not observed in healthy individuals (HC).

INDUSTRIAL APPLICABILITY

The present invention provides methods for predicting the effect of an IL-6 inhibitor in treating IL-6- and neutrophil-associated diseases by using the expression levels of neutrophil-associated genes of patients with the disease as an indicator. The present invention further provides novel methods for treating patients with IL-6- and neutrophil-associated diseases whose expression levels of neutrophil-associated genes are high. By the present invention, administration of an IL-6 inhibitor to patients for whom therapeutic effect by an IL-6 inhibitor cannot be expected or who will be forced to experience serious side effects or worsening of concomitant immunological abnormalities can be avoided, and appropriate therapeutic methods can be selected. 

1. A method for treating an IL-6 associated disease, comprising administering an IL-6 inhibitor to a patient having the IL-6 associated disease wherein a sample obtained from the patient prior to the administration is determined to have a high expression level of a neutrophil-associated gene compared to a control.
 2. The method of claim 1, wherein the control is the expression level of the neutrophil-associated gene in a sample obtained from a healthy individual.
 3. A method for treating an IL-6 associated disease, comprising administering an IL-6 inhibitor to a patient for whom prior treatment of the IL-6 inhibitor has been determined to be highly effective, wherein the prior treatment is determined to be highly effective by: (i) measuring the expression level of a neutrophil-associated gene in a sample obtained from the patient with an IL-6- associated disease who has been administered the IL-6 inhibitor, and (ii) comparing the expression level measured in (i) with a control, wherein the therapeutic effect of the IL-6 inhibitor is indicated to be high when the expression level of the neutrophil-associated gene is lower than the control.
 4. The method of claim 3, wherein the control is the expression level of the neutrophil-associated gene in a sample obtained from the patient prior to administration of the IL-6 inhibitor.
 5. The method of claim 1, wherein the neutrophil-associated gene is at least one gene selected from the group consisting of CTSG (cathepsin G), DEFA4 (defensin alpha 4, corticostatin), AZU1 (azurocidin 1), BPI (bactericidal/permeability-increasing protein), ELANE (elastase, neutrophil expressed), LTF (lactotransferrin), DEFA3 (defensin alpha 3, neutrophil-specific), LCN2 (lipocalin 2), and CAMP (cathelicidin antimicrobial peptide).
 6. The method of claim 1, wherein the IL-6 inhibitor is an anti-IL-6 receptor antibody. 7-10. (canceled)
 11. The method of claim 6, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 12. The method of claim 1, wherein the determined expression level of the neutrophil-associated gene is five times or higher, or ten times or higher, than the expression level of the neutrophil-associated gene in a sample obtained from a healthy individual.
 13. The method of claim 1, wherein the IL-6-associated disease is neuromyelitis optica, Neuro-Sweet disease, stroke, or cerebral infarction.
 14. The method of claim 3, wherein the neutrophil-associated gene is at least one gene selected from the group consisting of CTSG (cathepsin G), DEFA4 (defensin alpha 4, corticostatin), AZU1 (azurocidin 1), BPI (bactericidal/permeability-increasing protein), ELANE (elastase, neutrophil expressed), LTF (lactotransferrin), DEFA3 (defensin alpha 3, neutrophil-specific), LCN2 (lipocalin 2), and CAMP (cathelicidin antimicrobial peptide).
 15. The method of claim 3, wherein the IL-6 inhibitor is an anti-IL-6 receptor antibody.
 16. The method of claim 15, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 17. The method of claim 3, wherein the measured expression level of the neutrophil-associated gene in (i) is less than 0.5 times or less than 0.2 times the expression level of the neutrophil-associated gene in a sample obtained from the patient before the prior administration of an IL-6 inhibitor.
 18. The method of claim 3, wherein the IL-6-associated disease is neuromyelitis optica, Neuro-Sweet disease, stroke, or cerebral infarction.
 19. The method of claim 5, wherein the IL-6 inhibitor is an anti-IL-6 receptor antibody.
 20. The method of claim 19, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 21. A method for treating an IL-6 associated disease comprising administering an IL-6 inhibitor to a patient for whom treatment with an IL-6 inhibitor is predicted to be highly effective, wherein the treatment is predicted to be highly effective by: (i) measuring the expression level of a neutrophil-associated gene in a sample obtained from the patient with an IL-6 associated disease, and (ii) comparing the expression level measured in (i) with a control, wherein the therapeutic effect of the IL-6 inhibitor is indicated to be high when the expression level is higher than the control.
 22. The method of claim 21, wherein the control is the expression level of the neutrophil-associated gene in a sample obtained from a healthy individual.
 23. The method of claim 21, wherein the neutrophil-associated gene is at least one gene selected from the group consisting of CTSG (cathepsin G), DEFA4 (defensin alpha 4, corticostatin), AZU1 (azurocidin 1), BPI (bactericidal/permeability-increasing protein), ELANE (elastase, neutrophil expressed), LTF (lactotransferrin), DEFA3 (defensin alpha 3, neutrophil-specific), LCN2 (lipocalin 2), and CAMP (cathelicidin antimicrobial peptide).
 24. The method of claim 21, wherein the IL-6 inhibitor is an anti-IL-6 receptor antibody.
 25. The method of claim 24, wherein the anti-IL-6 receptor antibody is a chimeric antibody, a humanized antibody, or a human antibody.
 26. The method of claim 21, wherein the measured expression level of the neutrophil-associated gene is five times or higher, or ten times or higher, than the expression level of the neutrophil-associated gene in a sample obtained from the control.
 27. The method of claim 21, wherein the IL-6-associated disease is neuromyelitis optica, Neuro-Sweet disease, stroke, or cerebral infarction.
 28. A kit for predicting or determining the therapeutic effect of an IL-6 inhibitor in a patient suffering from an IL-6-associated disease, which comprises: (i) a reagent for measuring the expression level of at least one neutrophil-associated gene selected from the group consisting of CTSG (cathepsin G), DEFA4 (defensin alpha 4, corticostatin), AZU1 (azurocidin 1), BPI (bactericidal/permeability-increasing protein), ELANE (elastase, neutrophil expressed), LTF (lactotransferrin), DEFA3 (defensin alpha 3, neutrophil-specific), LCN2 (lipocalin 2), and CAMP (cathelicidin antimicrobial peptide); and (ii) a positive control sample for the expression level of the neutrophil-associated gene. 